title: Huang-Lian-Jie-Du-Decotion induced protective autophagy against the injury of cerebral ischemia/reperfusion via MAPK-mTOR signaling pathway.
abstract: BACKGROUND
  Huang-Lian-Jie-Du-Decotion (HLJDD, Hwangryun-Hae-Dok-Decotion in Japan), an ancient antipyretic and detoxifying traditional Chinese medicine formula, was reported to have protective effect on ischemic stroke.
  
  
  OBJECTIVE
  To investigate the therapeutic effect of HLJDD on ischemic stroke and explore its mode of action.
  
  
  METHODS
  A model of ischemic stroke in the rat was established after transient middle cerebral artery occlusion (MCAO) followed by reperfusion. Rats were assigned randomly to groups of control, sham, transient ischemia/reperfusion (I/R), and three treatment groups by HLJDD at 2.5, 5.0, 10.0mg/kg. The neurological deficit, the cerebral infarct size, morphology abnormality, biochemical parameters were examined, and the levels of relevant proteins were determined by immunoblotting analysis to evaluate the protective effects of HLJDD on ischemic stroke and explore the underlying mechanism.
  
  
  RESULTS
  Compared with I/R group, HLJDD significantly ameliorated neurological deficit and histopathology changes, decreased infarct area, and restored the levels of biochemical indicators including nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), glutathione disulfide (GSSG), total superoxide dismutase (T-SOD), Cu/Zn-SOD, Mn-SOD and glutathione peroxidase (GSH-PX). HLJDD also notably elevated the levels of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, and other autophagy related genes (Atgs), promoted the activation of extracellular signal-regulated kinases (ERK), protein kinase B (Akt), 3-phosphoinositide-dependent kinase (PDK1), and inhibited the activation of mammalian target of rapamycin (mTOR), c-Jun N-terminal protein kinases (JNK), p38, phosphatase and tensin homolog (PTEN).
  
  
  CONCLUSIONS
  HLJDD showed neuroprotective effects on ischemic stroke, at least in part to the induced protective autophagy via the regulation of mitogen-activated protein kinase (MAPK) signals. This Akt-independent protective autophagy is favorable in the treatment of stroke, avoiding unfavorable side-effects associated with the inactivation of Akt. The efficacy of HLJDD on ischemic stroke and its safety warranted by its long-term clinical use in traditional Chinese medicine favored further study to develop HLJDD as an effective therapeutic agent to treat ischemic stroke.
authors.affiliation: State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China.
authors.forename: Peng-Ran
authors.initials: PR
authors.lastname: Wang
authors.forename: Jun-Song
authors.initials: JS
authors.lastname: Wang
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Xing-Fang
authors.initials: XF
authors.lastname: Song
authors.forename: Na
authors.initials: N
authors.lastname: Tian
authors.forename: Ling-Yi
authors.initials: LY
authors.lastname: Kong
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000008:administration & dosage / Q000627:therapeutic use*; D001343:Autophagy / Q000187:drug effects*; D001522:Behavior, Animal / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000008:administration & dosage / Q000302:isolation & purification / Q000627:therapeutic use*; D020834:Ethnopharmacology; D002546:Ischemic Attack, Transient / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D015394:Molecular Structure; D018384:Oxidative Stress / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D004365:Drugs, Chinese Herbal; C061676:oren gedoku to; C546845:mTOR protein, rat; D058570:TOR Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases
keywords: (β-Nicotinamide adenine dinucleotide, reduced disodium salt, trihydrate); 2,3,5-triphenyltetrazolium chloride; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; 3-phosphoinositide-dependent kinase; 5,5′-Dithiobis-(2-nitrobenzoic acid); ANOVA; Akt; Atg; Autophagy; Autophagy related genes; BCA; BSA; Bicinchoninic acid; CCA; CMC-Na; DTNB; ECA; ECL; ERK; Fetal Bovine Serum Albumin; Fig.; Figure; GSH; GSH-PX; GSSG; HE; HLJDD; Huang-Lian-Jie-Du-Decotion; Huanglian-Jie-Du-Decotion; I/R; ICA; IPC; JNK; LC3; LCBF; LDF; MAPK; MCA; MCAO; MDA; Middle cerebral artery occlusion (MCAO); Mitogen-activated protein kinase (MAPK); NADH; NO; PAPTOR; PDK1; PI3K; PIP(2); PIP(3); PTEN; Protein kinase B (Akt); RICTOR; ROS; S.D.; S6K; SDS-PAGE; Stroke; T-SOD; TBS-T; TTC; WST-1; c-Jun N-terminal protein kinases; carboxymethyl cellulose sodium salt; common carotid artery; enhanced chemiluminescence; external carotid artery; extracellular signal-regulated kinases; glutathione; glutathione disulfide; glutathione peroxidase; hematoxylin–eosin; i.g.; internal carotid artery; intragastric administration; ischemia/reperfusion; ischemic preconditioning; laser Doppler flow meter; local cortical blood flow; mLST8; mLST8 and mammalian stress-activated protein kinase interacting protein; mSIN1; mTOR; mTOR complex1; mTORC1; malondialdehyde; mammalian lethal with Sec13 protein 8; mammalian target of rapamycin; microtubule-associated protein1 light chain 3; middle cerebral artery; middle cerebral artery occlusion; mitogen-activated protein kinase; nitric oxide; one-way analysis of variance; p70 ribosomal S6 kinase; phosphatidylinositol (3,4,5) trisphosphate; phosphatidylinositol (4,5) bisphosphate; phosphatidylinositol 3-kinase; protein kinase B; reactive oxygen species; scaffolding protein rapamycin-insensitive companion of mTOR; scaffolding protein regulatory-associated protein of mTOR; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; standard deviation; t-PA; tensin homolog; tissue-plasminogen activator; total superoxide dismutase; tris-buffered saline containing 0.1% Tween-20; v: v; volume: volume; w/v; weigh/volume